Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum
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Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC–MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.
KeywordsBrassica napus Phytohormones Salicylic acid Salicylic acid glucoside Verticillium longisporum Xylem sap
Days post inoculation
Salicylic acid glucoside
Salicylic acid (SA) is a phytohormone involved in the control of plant defence against pathogens. The role of SA in the activation of systemic acquired resistance (SAR) has been proven on biochemical and genetic levels. Treatment with SA or its analogues enhances the resistance of plants against pathogens, while mutants with disrupted SA synthesis and plants engineered to hydroxylate SA enzymatically exhibit increased susceptibility to diseases (reviewed by Loake and Grant 2007). Conversely, plants expressing bacterial genes for SA synthesis acquired enhanced resistance to pathogen infection (Verberne et al. 2000). While the role of SA in SAR is undisputed, SA is not the primary factor that is translocated from the place of infection to distant organs where it triggers SAR (Rasmussen et al. 1991; Vernooij et al. 1994). The identity of the primary systemic signal has not yet been conclusively established.
The major nonvolatile conjugate of SA is glucoside (SAG). SAG is regarded as a storage form or detoxification product of SA. Accumulation of SAG in the vacuole (Dean et al. 2005), and the absence of SAG from the phloem sap of plants with induced SAR (Enyedi et al. 1992), supported this view. Further corroboration came from the observation of a slow, long-lasting defence-like response in tobacco suspension culture treated by SAG, which was explained by a gradual release of SA by SAG hydrolysis (Kawano et al. 2004). This view was challenged recently by the work of Umemura et al. (2008), who used RNAi-mediated silencing of the glucosyl transferase responsible for SAG formation and showed that the activity contributed significantly to SAR development. The authors suggested that glucosylation of SA may actually be a key mediator of SAR. Overexpression of SA glucosyl transferase in Arabidopsis led to contradictory results, since the levels of free SA and SAG (as well as the glucose ester of SA) decreased rather than increased (Song et al. 2008).
Salicylic acid derivatives can be transported over long distances principally via phloem or xylem. Phloem moves plant metabolites in the source-to-sink direction while xylem transport takes place along the transpiration stream. Solutes can thus be transported in opposite directions in xylem and phloem, depending on the ratio of photosynthetic and respiratory activities of the organs involved. In phloem, SA levels are elevated upon pathogen attack (Métraux et al. 1990; Yalpani et al. 1991). In cucumber, free SA is translocated by phloem from cotyledon to leaves (Mölders et al. 1996), but phloem transport of SA from an inoculated leaf is low (Rasmussen et al. 1991). Conjugated forms of SA were not found in phloem sap of infected tobacco plants (Enyedi et al. 1992). In contrast to phloem, until recently, only indirect evidence indicated that xylem may be involved in the transport of SA. The distribution of [14C]-SA from an infiltrated leaf within the Arabidopsis rosette could not be explained by phloem translocation alone (Kiefer and Slusarenko 2003). Rocher et al. (2006) applied [14C]-labelled SA exogenously to the cotyledons of castor bean and observed movement of radioactivity via phloem towards the basal part of the seedling, where part of it was transported into the xylem and carried back to the shoot. The authors suggested that long-distance transport of SA via xylem may contribute to SA accumulation in distal parts of the plant in response to infection of its lower parts, in addition to endogenous synthesis and allocation by phloem. Their attempts to determine endogenous SA in xylem sap of castor bean seedlings failed.
Verticillium longisporum is a soil-borne fungal pathogen with host specificity for the genus Brassica. The fungus enters host plants through their roots and colonises xylem vessels, to which it remains restricted for most its infection phase (Eynck et al. 2007; Zhou et al. 2006). The main motivation for the taxonomical separation of V. longisporum from the closely related V. dahliae is the ability of V. longisporum to infect crucifers. V. dahliae is therefore commonly used in studies of V. longisporum pathogenicity as a control to test for the specificity of the observed effects.
In this work we demonstrate that SA and SAG are present in xylem sap of Brassica napus plants, and show that the concentrations of both compounds increase following infection with Verticillium longisporum.
Materials and methods
Rapid-cycling rape (Brassica napus var. napus, genome ACaacc) was originally provided by P.H. Williams (Department of Plant Biology, University of Wisconsin-Madison, WI; Crucifer Genetics Cooperative, Stock number 5). Seeds were surface sterilised by immersing them in 70% ethanol for 30 s and then rinsing with sterilised tap water for 30 s three times.
Verticillium longisporum VL 43 from B. napus and V. dahliae VD 73 from Linum usitatissimum were used in this study. Both strains were isolated from plants grown in northern Germany as described in Zeise and von Tiedemann (2001).
Fungal stocks with 1–3 × 106 conidia ml−1 were stored at −80°C in 25% glycerol.
Preparation of inoculum for plant infection
Shake cultures were started by adding 500 μl spore suspension (1–3 × 106 conidia ml−1) to 300 ml Czapek Dox broth. The inoculated broth was incubated at 23°C on a rotary shaker at 100 rpm in the dark. After 10–14 days, the culture was filtered through sterile gauze. Spore concentration was determined using a haemocytometer and diluted to 1 × 106 spores ml−1.
Plant inoculation and cultivation
Rapid-cycling rapeseeds were germinated in autoclaved sand in climate-controlled chambers (30 kLux, 60% humidity, 22/20°C day/night, and 15-h day length; Philips TL5 HO lamps). Seven-day-old seedlings were inoculated by root-dipping in a spore suspension of either V. longisporum (isolate 43, 1 × 106 spores ml−1) or V. dahliae (isolate 73, 1 × 106 spores ml−1) for 30–45 min. As a control, seedlings were dipped in sterile water. After inoculation, single seedlings were potted in a sterile sand–soil mixture (1:1) and grown under the conditions described above.
Xylem sap preparation
Rapeseed plants were harvested 28 days post inoculation (dpi). For time-course experiments, xylem sap was collected at 14, 21, 28, and 35 dpi. Shoots were cut above the hypocotyl, and xylem sap was collected by pressurizing roots to 4 × 105 Pa for 15 min using a Scholander pressure chamber (Scholander et al. 1965). Xylem sap was filtered through a 0.2-μm-Teflon filter (WICOM, Heppenheim, Germany) and subjected to HPLC–MS analysis immediately or stored at −20°C. Xylem sap was checked for contamination with cytoplasm by determining malate dehydrogenase activity (absence of activity indicated absence of cytoplasmic contamination).
Determination of sucrose concentration in xylem sap
The concentration of total sucrose and glucose in xylem sap was determined using the assay kit Enzytec fluid sucrose (r-biopharm, Darmstadt, Germany), which is based on enzymatic hydrolysis of sucrose, phosphorylation of glucose by the activity of hexokinase and oxidation of glucose-6-phosphate to gluconate-6-phosphate by glucose-6-phosphate dehydrogenase. The increase in NADH concentration was monitored by UV absorption at 340 nm (Specord 40, Analytik Jena, Germany). The concentration of free glucose was determined using an Enzytec fluid d-Glucose assay kit (r-biopharm, Darmstadt, Germany), which is based on the same principle except for the omission of the sucrose hydrolysis step. Standard deviation of technical replicas was lower than 4% for both assays. The concentration of sucrose was determined by subtracting the glucose concentration from the sum of sucrose and glucose.
Preparation of shoot extracts
The shoots of plants from which xylem sap was obtained were shock frozen with liquid nitrogen and stored at −80°C. Frozen shoot material was lyophilized (at −20°C) and ground in a ball mill. Extraction was performed as follows: to 50 mg dry shoot material (two replications for each plant), 1.5 ml methanol (gradient quality; Fisher Scientific, Schwerte, Germany) and 1.0 ml chloroform (p.a.; Roth, Karlsruhe, Germany) were added. The mixture was shaken vigorously and incubated for 5 min at 37°C. Subsequently, 2 ml either twice-distilled water or 2% formic acid (puriss. p.a.; Fluka, Seelze, Germany) in water were added. The suspension was shaken for 2 h at room temperature. The samples were centrifuged at 3,583 g for 15 min, the phases were separated, and the polar phase dried under vacuum at 30°C. The residue was dissolved in 1 ml methanol/bi-distilled water (1:9), and the solution was filtered through a 0.2-μm-Teflon filter. Samples were directly subjected to HPLC–mass spectrometry (HPLC–MS) or stored at −20°C.
High performance liquid chromatography and mass spectrometry
Methanol (gradient quality; Fisher Scientific, Schwerte, Germany), acetonitrile (gradient quality; VWR, Darmstadt, Germany), and acetic acid (p.a.; Fluka) were used for the mobile phases.
For full-scan analysis, an HPLC system equipped with autosampler (ProStar 410, Varian, Darmstadt, Germany), binary pump system (ProStar 210, Varian), degasser, and a column oven coupled to an ion trap mass spectrometer (500-MS, Varian) via electrospray ionisation source was used. Samples were injected onto a reversed-phase column Polaris C18-Ether (100 × 2 mm, 3 μm particle size; Varian) kept at 40°C. The mobile phase consisted of a binary gradient of 7 mM acetic acid in 95% water with 5% acetonitrile (A), and 7 mM acetic acid in methanol (B): 0.0–2.2 min 10% B, 2.2–25.0 min from 10% B to 98% B, 25–30 min 98% B, followed by washing (98% B) and re-equilibration (10% B) steps at a flow rate of 0.2 ml/min. The electrospray ionisation source was operated either with negative or positive spray polarity with the mass spectrometer operating in a full scan mode at a mass range of a mass-to-charge ratio (m/z) 100–1,000 and a scan speed of 5,000 Da/s. Raw data files were converted into NetCDF format and processed using XCMS Analyte Profiling Software (Smith et al. 2006).
Phytohormones, SA, and SAG were quantified using multiple reaction monitoring mode on the HPLC system specified above connected to a triple-quadrupole mass spectrometer (1,200 L, Varian) with an electrospray interface. Standards of SA, jasmonic acid (JA), and abscisic acid (ABA) were purchased from Sigma-Aldrich. Salicylic acid glucoside was kindly provided by H. Kneifel (Research Centre Jülich, Institute Phytosphere, Jülich, Germany). The analytical compounds were separated on a reversed-phase column (Polaris C18-A, 125 × 2 mm, 5 μm particle size, Varian) at 40°C and eluted isocratically with 7 mM acetic acid in 47.5% water, 2.5% acetonitrile, and 50% methanol at a flow rate of 0.2 ml/min. The eluate was subjected to negative electrospray ionization, and the ions were detected using the following mass transitions: SAG m/z 298.8 → m/z 137 and m/z 298.8 → m/z 93; SA m/z 136.8 → m/z 93; ABA m/z 262.8 → m/z 153, JA m/z 208.9 → m/z 59.
Quantification of V. longisporum DNA
Fungal biomass was determined in lyophilized hypocotyls from single plants harvested at 28 dpi. DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method as modified by Brandfass and Karlovsky (2006), except for the final precipitation with polyethylene glycol (polyethylene glycol 6000, Serva, Heidelberg, Germany), which was performed as follows. After chloroform extraction, 700 μl DNA solution in CTAB buffer was transferred to a 1.5-ml tube containing 200 μl polyethylene glycol (30%) and 100 μl 5 M NaCl; the solution was mixed, incubated for 20 min at room temperature, and centrifuged for 15 min at 14,000g. The pellet was washed with 70% (v/v) ethanol, dried, and dissolved in 30 μl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). DNA concentration was estimated by densitometry after electrophoresis in 0.8% (w/v) agarose gels (Cambrex, Rockland, ME) in TAE buffer (40 mM Tris, 1 mM EDTA, pH 8.5) at 7 V/cm for 60 min. Densitometry was performed as described previously (Brandfass and Karlovsky 2006). Fungal DNA was quantified by real-time PCR as described by Eynck et al. (2007).
Data are presented as mean ± standard deviation. The standard deviations for the increases in SA or SAG relative to the control were calculated according to the error propagation formula. Differences in concentrations of phytohormones and SA derivatives among uninfected, V. longisporum-infected, and V. dahliae-infected plants were determined by ANOVA, and the significance of correlation coefficients was determined with 1-tailed t tests (SPSS 15.0; SPSS, Chicago, IL). All correlation coefficients presented in the figures are significant at the 0.05 level.
Salicylic acid glucoside in xylem sap and shoots of B. napus
Xylem sap extracted from B. napus plants infected with V. longisporum and from healthy controls was analysed by HPLC–MS to identify metabolites affected by the infection. Plants at 28 dpi were used because at this time the amount of xylem sap extractable from a single plant exceeded 100 μl. Xylem sap was filtered and subjected to HPLC–MS analysis directly.
The concentration of the sum of sucrose and glucose in xylem sap was determined in order to estimate the contamination of xylem sap with phloem sap. Xylem sap of infected plants contained 2.17 ± 1.04 mM sucrose plus glucose, and xylem sap of uninfected controls contained 3.10 ± 0.31 mM sucrose plus glucose, indicating that contamination of xylem with phloem sap was lower than 0.3% (see “Discussion” for calculation).
Comparison of the metabolic profiles of infected and healthy plants revealed a number of signals with intensities that were increased in infected plants. One of these signals had m/z = 299—the same as that of the molecular ion of SAG in negative ionisation mode.
Relationship between SA and SAG concentrations, disease symptoms and fungal biomass
Changes in concentrations of SA, SAG, JA, and ABA in xylem sap during infection of B. napus with V. longisporum
The levels of JA and ABA in xylem sap increased with plant age, but did not differ significantly between V. longisporum-infected, V. dahliae-infected and healthy plants (Fig. 5c, d).
We detected SA and SAG in xylem sap of B. napus and showed that their concentrations increased after infection with V. longisporum. High concentrations of SA occur in phloem sap after pathogen attack (Métraux et al. 1990; Rasmussen et al. 1991) but neither free endogenous SA nor its derivatives have been reported from xylem sap. A recent study of the re-distribution of [14C]-labelled SA (Rocher et al. 2006) inspired a hypothesis that the transpiration stream contributes to long-distance transport of SA in plants, but an attempt to detect SA in xylem sap by these latter authors failed.
In principle, the SA and SAG found in xylem sap could have originated from contamination with other tissues. Pollution with cytoplasm from damaged cells appeared negligible because activity of malate dehydrogenase was undetectable, but contamination with phloem sap cannot be excluded. Sucrose is a biomarker for phloem; while present in only trace quantities in xylem sap (Nakamura et al. 2008), sucrose concentrations of 0.98 and 1.34 M were reported for phloem sap of B. napus (Lohaus and Moellers 2000). Comparison of these values with our results (2.17 ± 1.04 mM of sucrose plus glucose for infected plants, 3.10 ± 0.31 mM for uninfected controls) indicates that xylem sap contained less then 0.3% phloem sap. The concentration of free glucose in xylem sap was 1.31 ± 0.53 mM (controls) and 1.30 ± 0.08 mM (infected plants), while Nakamura et al. (2008) reported 0.30 mM. Since we assumed that all glucose in xylem originated from hydrolysed sucrose, the contamination of xylem sap with phloem sap was probably even lower than the estimate given above.
Salicylic acid glucoside is formed by glucosyl transferase in a concentration-dependent manner (Dean et al. 2005). Because glycosylation is an intracellular process, SAG must be transported into xylem from the surrounding tissues. Similar ratios of SAG and SA in shoot tissue and in xylem sap (Fig. 2a, b) indicate that both metabolites are secreted into xylem with the same efficiency, contrasting with the selective transport of SAG into the vacuole (Dean and Mills 2004). Recent findings that the suppression of SA glycosylation in rice by gene silencing reduced the development of resistance (Umemura et al. 2008) indicates that SAG may play a central role in SAR. Overexpression of SA glucosyl transferase in Arabidopsis, however, increased susceptibility to a bacterial pathogen (Song et al. 2008). This contradiction was resolved by finding that SA and SAG in transgenic plants decreased rather than increased due to increased activity of SA methyl transferase (Song et al. 2008).
Many biologically active secondary metabolites synthesised in the roots are transported acropetally with the transpiration stream (Satoh 2006). Abscisic acid is synthesised during drought stress in roots and transported to the shoots, where it triggers a drought response (Hartung et al. 2002). Zeatins are also carried from roots to shoots (Kato et al. 2002; Kuroha et al. 2005). According to our data, in B. napus, SA and SAG are transported in the same manner. Our results proved Rocher et al. (2006) right in their prediction that “endogenous SA levels in xylem sap should be far from negligible in infected plants”. In this context, it is interesting to note that the specific activity of SA glucosyl transferase in oat was ten-times higher in roots than in shoots (Yalpani et al. 1992).
In A. thaliana, the role of the SA pathway in response to Verticillium infection has been studied by Veronese et al. (2003), Tjamos et al. (2005) and Johansson et al. (2006). The first two groups worked with V. dahliae and obtained contradictory results regarding the expression of SA-dependent pathogenesis-related genes. Because the taxonomical status of V. longisporum is controversial, and its separation from V. dahliae is sometimes difficult, one of the strains used in these studies might actually have been V. longisporum (A. von Tiedemann, unpublished results). The third laboratory worked with V. longisporum and described the induction of PR-1 and PR-2 as early as 7 dpi, but the SA pathway was found not to be involved in the defence because plants with defective SA pathway did not exhibit enhanced susceptibility. In our experiments, the amount of SAG in the shoot was correlated with fungal biomass in the hypocotyl (Fig. 4a). Similar correlations were found for SA in shoot extracts and fungal DNA, and for SA and SAG in xylem sap and DNA (data not shown). Furthermore, both SA and SAG concentrations were correlated negatively with shoot length (Fig. 3). The determination of SA and SAG concentrations in A. thaliana after infection might help assess the suitability of the A. thaliana/V. longisporum pathosystem as a model for Verticillium disease of oilseed rape.
V. dahliae-infected plants were indistinguishable from uninfected controls both in SA and SAG levels and in their phenotype (Fig. 5a, b). The lack of disease symptoms in B. napus infected with V. dahliae has been demonstrated before by Eynck et al. (2007).
Johansson et al. (2006) demonstrated that the JA pathway was involved in the response of A. thaliana to V. longisporum, and that ABA was not required (although an ABA2 mutant was impaired in defence reaction). In our work with B. napus, JA levels in xylem sap increased, and those of SAG decreased with plant age (Fig. 5). This is indicative of an antagonistic relationship between the two pathways (Takahashi et al. 2004). Because infected and healthy plants exhibited similar changes in JA concentration over time, however, aging-related processes were more likely the cause. JA is known to be involved in senescence (Fukushige et al. 2002). Obviously the roles of JA in the defence of Arabidopsis and Brassica against V. longisporum are different, possibly reflecting the fact that Brassica, unlike Arabidopsis, is a natural host for the fungus.
Veronese et al. (2003) showed that ABA (but not SA or JA) acted as a regulator of the A. thaliana response to V. dahliae infection (the strain used might actually have been V. longisporum). Antagonism between the ABA and SA pathways was demonstrated in Arabidopsis (Yasuda et al. 2008), but in our system the increase in SA concentration caused by V. longisporum infection did not affect endogenous ABA levels (Fig. 5).
This work demonstrates for the first time the presence of SA and SAG in xylem sap. These two metabolites thus expand the list of phytohormones transported with the transpiration stream from roots to shoots (Satoh 2006). The biological role of the enhanced levels of SA and SAG in xylem sap after infection with a vascular pathogen remains to be elucidated.
This research work was part of an interfacultative research unit FOR546 funded by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany. We gratefully thank Prof. H. Kneifel (Forschungszentrum Jülich, Jülich, Germany) for providing us with the SAG standard. The technical assistance of P. Bartoschek and M. Bossmann is highly appreciated.
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